Short immunomodulatory oligonucleotides

ABSTRACT

The invention relates to modulation of the immune system. More particularly, the invention relates to modulating the immune system through the use of oligonucleotide-derived compounds. The invention provides immunostimulatory agents that are less expensive to make than existing immunostimulatory oligonucleotides. The immunostimulatory agents according to the invention can, in preferred embodiments, cause immune stimulation across species lines.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/361,111, filed on Feb. 7, 2003 (Now U.S. Pat. No. 7,354,907), thecontents of which are incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to modulation of the immune system. Moreparticularly, the invention relates to modulating the immune systemthrough the use of oligonucleotide-derived compounds.

2. Summary of the Related Art

Tokunaga et al, J. Natl. Cancer Inst. 72:955-962 (1984); Messina et al.,J. Immunol. 147: 1759-1764 (1991); Krieg et al., Nature 374: 546-549(1995); Sato et al, Science 273: 352-354 (1996), teach that the presenceof CpG dinucleotides in certain sequence contexts in bacterial andsynthetic oligodeoxyribonucleotides (CpG DNAs) are known to activatevertebrate innate immune reaction, T-cells and B cells.

Yamamoto et al., Jpn. J. Cancer Res. 79: 866-873 (1988); Halpern et al.,Cell Immunol., 167: 72-78 (1996); Klinman et al., Proc. Natl. Acad. Sci.U.S.A. 93: 2879-2883 (1996); Zhao et al., Antisense Nucleic Acid DrugDev. 7: 495-502 (1997) teach that the activation of immune cells by CpGDNA induces secretion of a number of cytokines, including IFN-γ, IL-12,TNF-α, and IL-6, and stimulates expression of costimulatory surfacemolecules.

Krieg et al., supra; Yamamoto et al, J. Immunol. 148; 4072-4076 (1992);Tokunaga et al., Microbiol. Immunol. 36: 55-66 (1992); Liang et al., J.Clin. Invest. 98: 1119-1129 (1996); Hartmann et al., J. Immunol. 164:1617-1624 (2000), teach that the presence of a CpG dinucleotide and thesequences flanking the dinucleotide play a critical role in determiningthe immunostimulatory activity of DNA, that CpG dinucleotides inpalindromic or non-palindromic hexameric sequences (P₁P₂CGP₃P₄) arerequired for immune stimulation, and further, that PuPuCGPyPy and PuTCGmotifs optimally activate murine and human immune systems, respectively.

While these findings demonstrate that oligonucleotides are useful asimmune stimulating agents, some problems with such use still exist. Forexample, long oligonucleotides are expensive to make and speciesspecificity of flanking sequences limits the breadth of utility of anygiven oligonucleotide. There is, therefore, a need for less expensiveimmunostimulatory agents, and preferably immunostimulatory agents thathave cross-species efficacy.

BRIEF SUMMARY OF THE INVENTION

The invention provides immunostimulatory agents that are less expensiveto make than existing immunostimulatory oligonucleotides. Theimmunostimulatory agents according to the invention can, in preferredembodiments, cause immune stimulation across species lines.Surprisingly, the present inventors have discovered that shortoligonucleotide-based agents that are linked together with appropriatelinkers can be made inexpensively and can be designed to cause immunestimulation in multiple species.

In a first aspect, the invention provides an immunomer comprising two ormore oligonucleotide branches linked together and having the structure:5′-N_(n)pYpRpN_(n)p3′-L_(m)-3′N_(n)pRpYpN_(n)-5′; wherein each N isindependently selected from a nucleoside, a nucleoside analog; anarabinonucleoside, or an abasic sugar; each p is independently a naturalor modified internucleoside linkage; at least one Y is selected from thegroup consisting of cytosine, 5-hydroxycytosine, N4-alkyl-cytosine,4-thiouracil or other non-natural pyrimidine nucleoside or2-oxo-7-deaza-8-methyl-purine, wherein when the base is2-oxo-7-deaza-8-methyl-purine, it is covalently bound to the 1′-positionof a pentose via the 1 position of the base; at least one R is selectedfrom the group consisting of guanine, 2-amino-6-oxo-7-deazapurine,2-amino-6-thiopurine, 6-oxopurine, or other non-natural purinenucleoside; L is a non-nucleotidic linker; each _(n) is independently anumber from 0-4, provided that neither branch exceeds 6 nucleotides; mis a number from 0-10 and wherein each N may optionally andindependently be covalently linked to a non-nucleotidic linker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows concentration-dependent cytokine induction in BALB/c mousespleen cell cultures (A) and the ratio of IL-12 to IL-6 induced inBALB/c (B), C57BL/6 (C), and CH3/HeJ (D) mice spleen cell cultures at 3μg/mL concentration of immunomers, 1 (squares), 2 (circles), 3(triangles) and 4 (reversed triangles).

FIG. 2 shows the effect of the nucleotide preceding CpG dinucleotide inshort immunomers as determined by the induction of IL-12 secretion inBALB/c mouse spleen and J774 macrophage cell cultures at a concentrationof 10.0 μg/mL of immunomers.

FIG. 3A shows a gel demonstrating activation of NF-κB in J774macrophages after stimulation for an hour with 10.0 μg/mL of immunomers.lane 1 is media treated control; lane 2 is human specific CpG DNA 2;lane 3 is control non-CpG DNA 5; lane 4 is mouse specific CpG DNA 1;lane 5 is LPS at 0.1 μg/mL; lane 6 is immunomer 3; and lane 7 isimmunomer 4.

FIG. 3B shows p38 phosphorylation in J774 macrophages followingactivation with immunomers for 30 minutes at 10.0 μg/mL concentration.Lane 1 is media treated control; lane 2 is mouse specific CpG DNA 1;lane 3 is immunomer 3; lane 4 is immunomer 4; lane 5 is human specificCpG DNA 2; and lane 6 is LPS at 0.1 μg/mL. Total p38 content is shown inlower panel.

FIG. 4 shows induction of IL12, IFN-γ, IL-6, and IL-10 secretion inhuman peripheral blood mononuclear cell (hPBMC) cultures at 1.0 μg/mLconcentration of short immunomers after 72 hr treatment.

FIG. 5A shows induction of IL-5 (top) and IFN-γ (bottom) secretion inconalbumin-sensitized AKR/J mice spleen cell cultures at 10.0 μg/mLconcentration of immunomers. A stands for allergen (conalbumin)senstized but not treated with immunomers.

FIG. 5B shows antitumor activity of immunomer 3 in nude mice bearingMCF-7 human breast cancer xenograft. Control represents the group ofmice treated with saline. * Represents statistically significant value(p<0.01).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to modulation of the immune system. Moreparticularly, the invention relates to modulating the immune systemthrough the use of oligonucleotide-derived compounds. The patents andpublications cited herein reflect the knowledge of those skilled in theart and are hereby incorporated by reference in their entirety. Anyconflict between the teachings of such references and the instantspecification shall be resolved in favor of the latter.

The invention provides immunostimulatory agents that are less expensiveto make than existing immunostimulatory oligonucleotides. Theimmunostimulatory agents according to the invention can, in preferredembodiments, cause immune stimulation across species lines.Surprisingly, the present inventors have discovered that shortoligonucleotide-based agents that are linked together with appropriatelinkers can be made inexpensively and can be designed to cause immunestimulation in multiple species.

In a first aspect, the invention provides an immunomer comprising two ormore oligonucleotide branches linked together and having the structure:5′-N_(n)pYpRpN_(n)p3′-L_(m)-3′N_(n)pRpYpN_(n)-5′; wherein each N isindependently selected from a nucleoside, a nucleoside analog; anarabinonucleoside, or an abasic sugar; each p is independently a naturalor modified internucleoside linkage; at least one Y is selected from thegroup consisting of cytosine, 5-hydroxycytosine, N4-alkyl-cytosine,4-thiouracil or other non-natural pyrimidine nucleoside or2-oxo-7-deaza-8-methyl-purine, wherein when the base is2-oxo-7-deaza-8-methyl-purine, it is covalently bound to the 1′-positionof a pentose via the 1 position of the base; at least one R is selectedfrom the group consisting of guanine, 2-amino-6-oxo-7-deazapurine,2-amino-6-thiopurine, 6-oxopurine, or other non-natural purinenucleoside; L is a non-nucleotidic linker; each _(n) is independently anumber from 0-4, provided that neither branch exceeds 6 nucleotides; mis a number from 0-10 and wherein each N may optionally andindependently be covalently linked to a non-nucleotidic linker.Preferred internucleoside linkages include phosphodiester,phosphorothioate and methylphosphonate linkages.

For purposes of the invention, a “non-nucleotidic linker” includes,without limitation a linker selected from a linker having a length offrom about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker,ethylene glycol linker, poly(ethylene glycol) linker,2-aminobutyl-1,3-propanediol linker, and branched alkyl linkers, acyclicalkyl linker, cyclic alkyl linker, aryl or heteroaryl linker,heterocyclic linker, polyalcohol linker, peptide linker, lipid linkerand carbohydrate linker, each of which may be substituted ornon-substituted.

For purposes of the invention, the term “oligonucleotide” refers to apolynucleoside formed from a plurality of linked nucleoside units. Sucholigonucleotides can be obtained from existing nucleic acid sources,including genomic or cDNA, but are preferably produced by syntheticmethods. In preferred embodiments each nucleoside unit includes aheterocyclic base and a pentofuranosyl, 2′-deoxypentfuranosyl,trehalose, arabinose, 2′-deoxy-2′-substituted arabinose,2′-O-substituted arabinose or hexose sugar group. The nucleosideresidues can be coupled to each other by any of the numerous knowninternucleoside linkages. Such internucleoside linkages include, withoutlimitation, phosphodiester, phosphorothioate, phosphorodithioate,alkylphosphonate, alkylphosphonothioate, phosphotriester,phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate,carbamate, morpholino, borano, thioether, bridged phosphoramidate,bridged methylene phosphonate, bridged phosphorothioate, and sulfoneinternucleoside linkages. The term “oligonucleotide” also encompassespolynucleosides having one or more stereospecific internucleosidelinkage (e.g., (Rp)— or (Sp)-phosphorothioate, alkylphosphonate, orphosphotriester linkages). As used herein, the terms “oligonucleotide”and “dinucleotide” are expressly intended to include polynucleosides anddinucleosides having any such internucleoside linkage, whether or notthe linkage comprises a phosphate group. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphorothioate, or phosphorodithioate linkages, or combinationsthereof.

The term “oligonucleotide” also encompasses polynucleosides havingadditional substituents including, without limitation, protein groups,lipophilic groups, intercalating agents, diamines, folic acid,cholesterol and adamantane. The term “oligonucleotide” also encompassesany other nucleobase containing polymer, including, without limitation,peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholino-backboneoligonucleotides, and oligonucleotides having backbone sections withalkyl linkers or amino linkers.

The oligonucleotides of the invention can include naturally occurringnucleosides, modified nucleosides, or mixtures thereof. As used herein,the term “modified nucleoside” is a nucleoside that includes a modifiedheterocyclic base, a modified sugar moiety, or a combination thereof. Insome embodiments, the modified nucleoside is a non-natural pyrimidine orpurine nucleoside, as herein described. In some embodiments, themodified nucleoside is a 2′-substituted ribonucleoside anarabinonucleoside or a 2′-deoxy-2′-substituted-arabinoside.

For purposes of the invention, the term “2′-substituted ribonucleoside”or “2′-substituted arabinoside” includes ribonucleosides orarabinonucleoside in which the hydroxyl group at the 2′ position of thepentose moiety is substituted to produce a 2′-substituted or2′-O-substituted ribonucleoside. Preferably, such substitution is with alower alkyl group containing 1-6 saturated or unsaturated carbon atoms,or with an aryl group having 6-10 carbon atoms, wherein such alkyl, oraryl group may be unsubstituted or may be substituted, e.g., with halo,hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carboalkoxy, or amino groups. Examples of 2′-O-substitutedribonucleosides or 2′-O-substituted-arabinosides include, withoutlimitation 2′-O-methylribonucleosides or 2′-O-methylarabinosides and2′-O-methoxyethylribonucleosides or 2′-O-methoxyethylarabinosides.

The term “2′-substituted ribonucleoside” or “2′-substituted arabinoside”also includes ribonucleosides or arabinonucleosides in which the2′-hydroxyl group is replaced with a lower alkyl group containing 1-6saturated or unsaturated carbon atoms, or with an amino or halo group.Examples of such 2′-substituted ribonucleosides or 2′-substitutedarabinosides include, without limitation, 2′-amino, 2′-fluoro, 2′-allyl,and 2′-propargyl ribonucleosides or arabinosides.

The term “oligonucleotide” includes hybrid and chimericoligonucleotides. A “chimeric oligonucleotide” is an oligonucleotidehaving more than one type of internucleoside linkage. One preferredexample of such a chimeric oligonucleotide is a chimeric oligonucleotidecomprising a phosphorothioate, phosphodiester or phosphorodithioateregion and non-ionic linkages such as alkylphosphonate oralkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat. Nos.5,635,377 and 5,366,878).

A “hybrid oligonucleotide” is an oligonucleotide having more than onetype of nucleoside. One preferred example of such a hybridoligonucleotide comprises a ribonucleotide or 2′-substitutedribonucleotide region, and a deoxyribonucleotide region (see, e.g.,Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

In a second aspect, the invention provides a method for modulating animmune response in a vertebrate. The method according to this aspect ofthe invention comprises administering to the vertebrate an immunomeraccording to the first aspect of the invention. For purposes of theinvention, the term “vertebrate” includes, without limitation, a fish,bird, or mammal. As used herein, the term “mammal” includes, withoutlimitation rats, mice, cats, dogs, horses, cattle, cows, pigs, rabbits,non-human primates, and humans. “Modulating an immune response” meanscausing an increase or decrease in, or activation of one or more ofB-cell induction, T-cell induction, cytokine induction, natural killercell induction, specific cell surface marker expression, chemokineinduction and activation of antigen presenting cells, such as dendriticcells, monocytes and macrophages.

In a third aspect, the invention provides a method for treating avertebrate having a disease. The method according to this aspect of theinvention comprises administering to the vertebrate an immunomeraccording to the first aspect of the invention. The term “vertebrate” isas described previously.

In the method according to this aspect of the invention, administrationof the immunomer or immunomer conjugate can be by any suitable route,including, without limitation, parenteral, oral, sublingual,transdermal, topical, intranasal, aerosol, intraocular, intratracheal,intrarectal, vaginal, by gene gun, dermal patch or in eye drop ormouthwash form. Administration of the therapeutic compositions ofimmunomers can be carried out using known procedures at dosages and forperiods of time effective to reduce symptoms or surrogate markers of thedisease. When administered systemically, the therapeutic composition ispreferably administered at a sufficient dosage to attain a blood levelof immunomer from about 0.0001 micromolar to about 10 micromolar. Forlocalized administration, much lower concentrations than this may beeffective, and much higher concentrations may be tolerated. Preferably,a total dosage of immunomer ranges from about 0.0001 mg per patient perday to about 200 mg per kg body weight per day. It may be desirable toadminister simultaneously, or sequentially a therapeutically effectiveamount of one or more of the therapeutic compositions of the inventionto an individual as a single treatment episode.

The following examples are provided to further illustrate certainparticularly preferred embodiments of the invention and are not intendedto limit the scope of the invention.

Example 1 Synthesis and Purification of Oligomers

CpG DNAs and immunomers were synthesized on a 1 to 2 μmole scale usingα-cyanoethylphosphoramidites on a PerSeptive Biosystem's 8990 ExpediteDNA synthesizer according to the manufacturer's directions. Thephosphoramidites of dA, dG, dC, and T were obtained from PE Biosystems(Foster City, Calif.). C3-linker phosphoramidite was obtained from GlenResearch Corporation (Sterling, Va.). Immunomers were synthesized onsolid supports carrying DiDMT protected glyceryl linker obtained fromChemGenes (Wilmington, Mass.) using a parallel synthesis. Beaucagereagent (R.I. Chemicals, Orange, Calif.) was used as an oxidant toobtain the phosphorothioate backbone modification. Immunomers weredeprotected using standard protocols, purified by HPLC, and dialyzedagainst USP quality sterile water for irrigation (Braun, Irving,Calif.). The immunomers were lyophilized and dissolved again indistilled water and the concentrations were determined from UVabsorbance at 260 nm. All immunomers were characterized by CGE andMALDI-TOF mass spectrometry (Applied Biosystem's Voyager-DE STRBiospectrometry Workstation, Foster City, Calif.) for purity andmolecular mass, respectively (Table 1). The purity of full-lengthimmunomers ranged from 89-95% with the rest being shorter by one or twonucleotides (n−1 and n−2) as determined by CGE and/or denaturing PAGE.All CpG DNAs contained less than 0.075 EU/mL of endotoxin as determinedby the Limulus assay (Bio-Whittaker, Walkersville, Md.).

TABLE 1 Sequences and chemical modifications of immunomers MolecularWeight No. Sequence^(a) Structure^(b) Length^(c) Found Calculated 1

18-mer 5705 5704 2

18-mer 5694 5695 3

6-mer 4308 4313 4

5-mer 3620 3623 5

6-mer 4310 4313 6

15-mer ND 4630 ^(a)Arrows indicate 5′→3′ directionality of CpGdinucleotide; chemical structures of X and Y linkers are shown below;^(b)Line drawings of immunomer structures, box represents the positionof CpG dinucleotide in the sequence, plain and spiked lines representnucleotide sequence and linker positions, respectively; ^(c)length ofeach segment in immumers excluding linkers.

Example 2 Cell Culture Conditions and Reagents

Spleen cells from 4-8 week old BALB/c, C57BL/6 or C3H/HeJ mice werecultured in RPMI complete medium under standard conditions. Murine J774macrophages (American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209) were cultured in Dulbecco'smodified Eagles medium supplemented with 10% (v/v) FCS and antibiotics(100 IU/mL of penicillin G/streptomycin). All other culture reagentswere purchased from Mediatech (Gaithersburg, Md.).

Example 3 PBMC Isolation from Fresh Murine and Human Blood

Peripheral blood mononuclear cells (PBMCs) from freshly drawn C57BL/6mouse or healthy human volunteer blood were isolated by Ficoll-Paquedensity gradient centrifugation method (Histopaque-1077, Sigma, St.Louis, Mo.) under standard conditions.

Example 4 Establishment of Th2 Immune Response in Mice

Four to six week old AKR/J male mice were obtained from Jackson Labs(Bar Harbor, Me.). The mice were given intraperitoneal injectionscontaining 200 μg of conalbumin (Sigma) with ImjectAlum adjuvant(Pierce, Rockford, Ill.) in 100 μL of PBS on days 0, 7, and intranasallychallenged on days 14, and 21. The mice were sacrificed 72 hr after thelast challenge by CO₂ inhalation. Spleens were excised and single cellsuspensions were prepared as described above. Spleen cells were treatedwith immunomers at different concentrations for 2 hr followed bytreatment with 50 μg/mL of conalbumin. Supernatants were harvested after72 hr and IL-5 and IFN-γ levels were measured by ELISA as describedbelow.

Example 5 Cytokine ELISAs

Mouse spleen or J774 cells were plated in 24-well dishes using 5×10⁶ or1×10⁶ cells/mL, respectively. The CpG DNA dissolved in TE buffer (10 mMTris-HCl, pH 7.5, 1 mM EDTA) was added to a final concentration of 0.03,0.1, 0.3, 1.0, 3.0, or 10.0 μg/mL to the cell cultures. The cells werethen incubated at 37° C. for 24 hr and the supernatants were collectedfor ELISA assays. The experiments were performed two or three times foreach CpG DNA in triplicate for each concentration. The secretion ofIL-12 and IL-6 was measured by conventional sandwich ELISA. The requiredreagents, including cytokine antibodies and standards were purchasedfrom PharMingen (San Jose, Calif.).

Example 6 Mouse Splenomegaly Assay

Female BALB/c mice (4-6 weeks, 19-21 gm) were divided into groups ofthree mice. CpG DNAs were dissolved in sterile PBS and administeredsubcutaneosly (SC) to mice at a dose of 5 mg/kg. The mice weresacrificed and the spleens were harvested and weighed.

Example 7 Preparation of J774 Cell Nuclear Extracts and EMSA

NF-κB activation in J774 cells treated with CpG DNAs was carried out andanalyzed by conventional EMSA (see e.g., Yu et al., Biochem. Biophys.Res. Commun. 297: 83-90 (2002).

Example 8 Preparation of J774 Cell Lysates and Western Blotting

To detect the phosphorylated and total p38 MAP kinase by Westernblotting, J774 cells were grown in serum-reduced medium (0.5% FCS) for24 hr and then stimulated with immunomers for 30 min. After stimulation,cells were washed with cold PBS and lysed in 2% SDS sample buffer as perthe protocol provided by Cell Signaling Technology (Beverly, Mass.).Crude lysates were resolved on 10% SDS polyacrylamide ReadyGels (BioRad,Hercules, Calif.) and blotted onto nitrocellulose membranes. Membraneswere probed with a phospho-p38 MAP kinase (Thr 180/Tyr 182) antibody andvisualized using enhanced chemiuminescence kit (PE Life Sciences,Boston, Mass.). The blots were then stripped and reprobed with anantibody to p38 MAPK that detects total levels of endogenous p38 MAPKprotein. All antibodies were purchased from Cell Signaling Technologies.

Example 9 In Vivo Nude Mice Model and Treatment Plan

The animal use and care protocols were approved by the InstitutionalCommittee on Animal Use and Care of the University of Alabama atBirmingham. Female athymic nude mice (nu/nu, 4-6 weeks old) wereobtained from Frederick Cancer Research and Development Center(Frederick, Md., USA) and inoculated with MCF-7 cells. The animalsbearing MCF-7 xenograft (50-100 mg) were randomly divided into varioustreatment groups and treated by subcutaneous injection withshort-immunomer 3 at a dose of 0.5 mg/kg or saline (control) on days 1,3, and 5 every week. The mice were monitored by general clinicalobservation as well as by body weight and tumor growth. Tumor growth wasrecorded with the use of calipers, by measuring the long and shortdiameters of the tumor. Tumor mass (in g) was calculated using theformula ½a×b², where “a” and “b” are the long and short diameters (incm), respectively.

Example 10 Activity of Short-Immunomers in Murine Spleen Cell and PBMCCultures

All immunomers showed a concentration-dependent induction of two typicalcytokines, IL-12 and IL-6, in BALB/c, C57BL/6, and C3H/HeJ mouse spleencell cultures. As expected, CpG DNA 1, containing a mouse specificsequence motif, generally showed greater activity than CpG DNA 2 with ahuman specific sequence motif (Table 2). At lower concentrations,immunomers 3 and 4 generally induced levels of cytokines lying betweenthose found with 1 and 2 in BALB/c mouse cells (FIG. 1). However, athigher concentrations the activities of 3 and 4 matched or exceeded thatof 1 even though the short immunomers do not contain the mouse specific‘GACGTT’ motif.

Levels of IL-12 and IL-6 induced by 1-5 at concentrations of 3.0 μg/mLin the three strains of mouse spleen cell cultures are shown in Table 2.Levels vary with mouse strain and depend on sequence (1 vs 2) and thestructure of the CpG DNA. Control immunomer 5 produced cytokine levelssimilar to background showing the need for the CpG dinucleotide. Ingeneral, a higher IL-12 to IL-6 ratio was found for short-immunomerscompared with CpG DNAs 1 and 2 (FIG. 1B). The results obtained in LPSnon-responsive strain, C3H/HeJ mouse spleen cell cultures suggest thatimmunomer activities are not due to LPS contamination acting on the TLR4receptor.

Previously, we showed that the presence of multiple CpG dinucleotides inoligonucleotides do not increase activity significantly over that due toa single copy. Here, control oligo 6 contains two copies of a CpGcontaining heptamer joined 5′→3′ through a C3-linker. Oligo 6 induced501±21 pg/mL of IL-12 and 514±164 pg/mL of IL-6 at a concentration of3.0 μg/mL in C57BL/6 mouse spleen cell culture assays compared with2833±341 and 2870±760 pg/mL of IL-12, and 24276±4740 and 14256±3304pg/mL of IL-6 by 1 and 3, respectively, at the same concentration. Theseresults confirm that the activity seen with 3 and 4 is not as a resultof the presence of two copies of CpG dinucleotide, but because of theiroptimal structure for receptor recognition.

In BALB/c mouse bone marrow derived dendritic cell and macrophage cellcultures, short-immunomer 3 induced similar levels of IL-12, IFN-γ, butsignificantly less IL-6, TNF-α and nitric oxide (NO) compared with CpGDNA 1 (data not shown).

Spleen cells consist of different subsets of cell population thanperipheral blood mononuclear cells (PBMCs). To examine whether thisdifference in cell population could result in different activity, weisolated PBMCs from C3H/HeJ mouse peripheral blood and tested for theability of short immunomers to induce IL-12 and IL-6 secretion. Theresults obtained at 10 μg/mL concentration of immunomers are presentedin Table 2. Immunomers 3 and 4 showed similar cytokine induction as inspleen cells.

TABLE 2 In vitro cytokine secretion in murine spleen cell, and PBMCcultures and in vivo spleen enlargement Spleen cell cultures^(c) PBMCcultures^(d) BALB/c C57BL/6 C3H/HeJ C3H/HeJ Spleen Wt. IL-12 IL-6 IL-12IL-6 IL-12 IL-6 IL-12 IL-6 No. (mg)^(b) (pg/mL) (pg/mL) (pg/mL) (pg/mL)(pg/mL) (pg/mL) (pg/mL) (pg/mL) 1 153 ± 10.0 1775 ± 139 9774 ± 798 889 ±71  7257 ± 132 931 ± 148 5465 ± 140 6541 ± 254 1602 ± 175 2 138 ± 10.3 612 ± 6 3211 ± 287 266 ± 12  3605 ± 404 157 ± 55 2099 ± 234 3838 ± 92 482 ± 93 3 146 ± 9.7 1532 ± 196 6086 ± 268 831 ± 114 11218 ± 631 650 ±89 3503 ± 53 2327 ± 373 1095 ± 106 4 125 ± 9.8 1711 ± 126 6606 ± 322 884± 45  8410 ± 309 783 ± 42 3456 ± 307 4400 ± 703  536 ± 26 5 NT  53 ± 4ND  77 ± 6  281 ± 57  24 ± 5 ND ND ND M/V^(a) 75 ± 2.4  47 ± 1   7 ± 1 71 ± 12  108 ± 38  24 ± 6  10 ± 3  25 ± 8 5 ± 3 ^(a)Medium or vehicle(PBS) control; ^(b)Average spleen weight of three mice per groupobtained after 48 hr of subcutaneous administration of single dose of 5mg/kg of immunomer; ^(c)at a concentration of 3.0 μg/mL of immunomer;^(d)at a concentration of 10.0 μg/mL of immunomer. NT and ND stand fornot tested and not detected, respectively.

Example 11 In Vivo Activity of Short Immunomers as Determined byIncrease in Spleen Weight or Splenomegaly

To further test the immunostimulatory activity, a single dose of 5 mg/kgof immunomers 1-4 was injected subcutaneously to BALB/c mice, and thespleen weights of mice (3 per group) were measured after 48 hrs[17,32,33]. Average weights of 153, 138, 146 and 125 mg (all±10 mg) werefound for 1-4, respectively, while PBS treated controls gave 75±6 mg.Treatment with 6 containing two copies of TCGTTGT gave 123±7 mg after 72hr recapitulating the lower activity of this compound in cell culture.These results further confirm the immunostimulatory activities found invitro.

Example 12 Influence of the Nucleotide Preceding CG Dinucleotide onImmunostimulatory Activity

Yu et al., Bioorg. Med. Chem. 11: 459-464 (2003) recently showed thatthe activity of the P₁P₂CGP₃P₄ motif containing an abasicdeoxynucleoside substitution at P₁ is influenced by the nature of thenucleoside (A, C, G, or T) present at P₂. Since we completely deleted P₁in short immunomers, we evaluated the effect of different nucleosides atP₂. Analogs of 3 were synthesized having A, G, or C, instead of T ateach 5′-end. In general, those with A, G, or C showed lower or noinduction of cytokines compared to 3 (IL-12 secretion in BALB/c spleenand J774 cell cultures is shown in FIG. 2).

Example 13 Short-Immunomers Activate NF-κB and p38 Map Kinase Pathwaysin J774 Cell Cultures

Stacey et al., J. Immunol. 157: 2116-2122 (1996) and Yi et al., J.Immunol. 161: 4493-4497 (1998) teach that CpG DNA activates the NF-κBand p38 MAP kinase signaling pathways, which play a critical role instimulating cytokine gene expression. To see if short immunomers work bythe same mechanism, we studied activation of NF-κB in J774 cell nuclearextracts (FIG. 3A). CpG DNA 1, which has a mouse specific sequence,activated NF-κB as expected (lane 4), as did LPS in lane 5 (FIG. 3A). Incontrast, CpG DNA 2, containing a human specific sequence, failed toinduce NF-κB (lane 2) suggesting specificity of the receptor for themouse specific CpG DNA sequence. Immunomers 3 and 4, which have two5′-accessible ends and five-nucleotide homology to the human specificmotif, GTCGTT, activated NF-κB (lanes 6 and 7) to the same extent as 1,suggesting that short immunomers are recognized by the mouse receptor.

Additionally, to examine the activation of stress kinase pathways byshort immunomers, we examined p38 MAP kinase activity in J774macrophages after treatment with immunomers (FIG. 3B). Both 3 and 4activated stress-activated pathways as shown by the presence ofphosphorylation product in J774 cell lysates within 30 min of treatment(lanes 3 and 4) as did 1 (lane 2) and LPS (lane 6). In contrast, 2containing human specific CpG motif failed to activate thestress-activated pathway. Consistent with the activation of NF-κB, 1, 3,and 4, but not 2 and 5, induced IL-12 and IL-6 in J774 cell cultures(data not shown).

Example 14 Immune-Response in Human PBMCs

Short-immunomers were further tested for their ability to stimulatehuman PBMCs to secrete cytokines IL-12, IL-6, IL-10, and IFN-γ.Representative data obtained in a single healthy donor PBMC cultures at1 μg/mL concentration are shown in FIG. 4. As expected, mouse specificCpG DNA 1 induced lower cytokine production than did human specific 2.Short-immunomers 3 and 4 gave similar or higher levels of cytokineinduction than did human specific CpG DNA 2. CpG DNA 6 showed lowercytokine induction than did 2, 3, and 4 despite containing as many CpGdinucleotides as 3 and 4. These results demonstrate that it is thestructure of the immunomer and not the number of CpG motifs that effectsthe immune stimulation.

Example 15 Effect of Short-Immunomer on IL-5 and IFN-1 Secretion inAllergen-Sensitized Spleen Cell Cultures

To assess the effects of treatment of short-immunomers on Th2 cytokinesassociated with allergic airway responses, we measured IL-5 and IFN-γsecreted in spleen cell cultures obtained from AKR/J mouse challengedwith conalbumin. In the absence of immunomer treatment,conalbumin-sensitized spleen cells secreted markedly higher levels ofIL-5 and low IFN-γ suggesting predominantly a Th2 type response (FIG.5A). When the allergen-primed spleen cells were treated with immunomers,a concentration-dependent decrease in IL-5 and increase in IFN-γsecretion was observed. FIG. 5A shows IL-5 (top plot) and IFN-γ (bottomplot) secretion levels at 10 μg/mL concentration of immunomers. Thesedata suggest that short-immunomers not only induce Th1 type cytokinesecretion but potently reverse Th2 responses in vitro, and shouldtherefore be useful as a potent adjuvant.

Example 16 Antitumor Activity of Short-Immunomer in Nude Mice BearingHuman Breast Cancer MCF-7 Xenograft

To examine if the potential in vitro activity of short-immunomers can betranslated in to in vivo antitumor activity, we administeredshort-immunomer 3 subcutaneously at a dose of 0.5 mg/kg three times aweek to nude mice bearing MCF-7 breast cancer xenografts that expresswild-type p53. At the relatively low dose, immunomer 3 gave 51%inhibition of MCF-7 tumor growth on day 24 compared with the salinecontrol (p<0.01) (FIG. 5B). These antitumor studies further suggest thatshort-immunomers exhibit potent antitumor activity in vivo as a resultof potent immune stimulation.

1. A method for modulating an immune response in a vertebrate,comprising administering to the vertebrate an immunomer comprising twoor more oligonucleotide branches linked together and having thestructure: 5′-N_(n)pYpRpN_(n)p3′-L_(m)-3′N_(n)pRpYpN_(n)-5′; whereineach N is independently selected from a nucleoside, a nucleoside analog;an arabinonucleoside, or an abasic sugar; each p is independently anatural or modified internucleoside linkage; at least one Y is selectedfrom the group consisting of cytosine, 5-hydroxycytosine,N4-alkyl-cytosine, 4-thiouracil or other non-natural pyrimidinenucleoside or 2-oxo-7-deaza-8-methyl-purine, wherein when the base is2-oxo-7-deaza-8-methyl-purine, it is covalently bound to the 1′-positionof a pentose via the 1 position of the base; at least one R is selectedfrom the group consisting of guanine, 2-amino-6-oxo-7-deazapurine,2-amino-6-thiopurine, 6-oxopurine, or other non-natural purinenucleoside; L is a non-nucleotidic linker; each _(n) is independently anumber from 0-4, provided that neither branch exceeds 6 nucleotides; mis a number from 0-10 and wherein each N may optionally andindependently be covalently linked to a non-nucleotidic linker.
 2. Themethod according to claim 1, wherein the vertebrate is selected from thegroup consisting of fish, birds, and mammals.
 3. The method according toclaim 2, wherein the mammal is selected from the group consisting ofrats, mice, cats, dogs, horses, cattle, cows, pigs, rabbits, non-humanprimates, and humans.
 4. A method for treating a vertebrate having adisease, comprising administering to the vertebrate an immunomercomprising two or more oligonucleotide branches linked together andhaving the structure: 5′-N_(n)pYpRpN_(n)p3′-L_(m)-3′N_(n)pRpYpN_(n)-5;wherein each N is independently selected from a nucleoside, a nucleosideanalog; an arabinonucleoside, or an abasic sugar; each p isindependently a natural or modified internucleoside linkage; at leastone Y is selected from the group consisting of cytosine,5-hydroxycytosine, N4-alkyl-cytosine, 4-thiouracil or other non-naturalpyrimidine nucleoside or 2-oxo-7-deaza-8-methyl-purine, wherein when thebase is 2-oxo-7-deaza-8-methyl-purine, it is covalently bound to the1′-position of a pentose via the 1 position of the base; at least one Ris selected from the group consisting of guanine,2-amino-6-oxo-7-deazapurine, 2-amino-6-thiopurine, 6-oxopurine, or othernon-natural purine nucleoside; L is a non-nucleotidic linker; each _(n)is independently a number from 0-4, provided that neither branch exceeds6 nucleotides; m is a number from 0-10 and wherein each N may optionallyand independently be covalently linked to a non-nucleotidic linker. 5.The method according to claim 4, wherein the vertebrate is selected fromthe group consisting of fish, birds, and mammals.
 6. The methodaccording to claim 5, wherein the mammal is selected from the groupconsisting of rats, mice, cats, dogs, horses, cattle, cows, pigs,rabbits, non-human primates, and humans.
 7. The method according toclaim 4, wherein the disease to be treated is selected from the groupconsisting of cancer, an autoimmune disorder, airway inflammation,asthma, allergy, and a disease caused by a pathogen.
 8. The methodaccording to claim 4, further comprising administering an agent selectedfrom the group consisting of vaccines, allergens, antigens, antibodies,monoclonal antibodies, chemotherapeutic drugs, antibiotics, lipids, DNAvaccines and adjuvants.